We used the Xenopus laevis oocyte expression system to characterize adenosine 3',5'-cyclic monophosphate (cAMP) activation of the cystic fibrosis transmembrane conductance regulator (CFTR). With conventional two-microelectrode voltage-clamp techniques, we recorded transmembrane conductance (G _m) and membrane current (I _m). Using five different sine wave frequencies, we also monitored changes of the plasma membrane surface area by recording continuously membrane capacitance (C _m) under voltage-clamp conditions. Impedance spectra recorded in the frequency range 0.1–500 Hz showed that, at least up to 200 Hz, C _m is independent of the frequency. In control oocytes, cAMP (100 µM) treatment did not affect G _m or I _m but evoked a small, slowly occurring increase in C _m, probably mediated by cAMP-stimulated exocytosis. However, in oocytes expressing CFTR, large simultaneous increases of G _m, I _m and C _m occurred after stimulation with cAMP. Oocytes injected with the ΔF508 CFTR mutant behaved like control oocytes and cAMP had no additional effects on G _m, I _m or C _m. In oocytes injected with wild-type CFTR, adenosine 5'-triphosphate (ATP, 100 µM) did not activate the cAMP-induced augmentation of I _m, G _m or C _m further. On the other hand, cAMP-induced increases in C _m were reduced significantly by the specific blockers of protein kinase A (PKA) KT5720 and N-[2-(methylamino-9-ethyl]-5-isoquinolinesulphonamide hydrochloride (H8), whereas the increases in G _m and I _m were essentially unaffected by these agents. Reducing intracellular Ca²⁺ by injection of a Ca²⁺ chelator 1,2-bis (2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) prevented PKA-dependent exocytosis while activation of I _m and G _m of already-inserted CFTR still could be detected. The specific cAMP antagonist adenosine 3',5'-cyclic monophosphothioate Rp diastereomer (RpcAMPS) completely suppressed the effects of cAMP on all parameters. These findings are consistent with the concept of different pathways of CFTR activation by cAMP: already-inserted CFTR Cl^– channels are activated directly by cAMP, while traffic of CFTR proteins from an intracellular pool to the plasma membrane and functional insertion into the plasma membrane occurs via cAMP- and Ca²⁺-dependent PKA-mediated exocytosis.